Biochem. J. (1994) 302, 781-790 (Printed in Great Britain)

Human inositol 1,4,5-trisphosphate type-1 receptor, InsP3Rl: structure,function, regulation of expression and chromosomal localizationNorihiko YAMADA,* Yasutaka MAKINO,t** Robert A. CLARK,: Doran W. PEARSON,t Marie-Genevieve MATTEI,§Jean-Louis GUENET,11 Eisaku OHAMA,T Ichiro FUJINO,* Atsushi MIYAWAKI,* Teiichi FURUICHI,*tt and Katsuhiko MIKOSHIBA*tt*Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan, tMinase Research Institute,Ono Pharmaceutical Co. Ltd., 3-1-1 Sakurai Shimamoto-cho, Mishima-gun, Osaka 618, Japan, IThe Division of Infectious Disease, College of Medicine,The University of Iowa, 200 Hawkins Drive, SW54 GH, Iowa City, IA 52242 U.S.A., §Unite de Recherche no. 242, Physiopathologie Chromosomique Hospital d'enfants,Groupe Hospitalier de la Timone, 13385 Marseille Cedex 5, France, llInstitut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France, ¶The Division of Neuropathology,Institute of Neurological Sciences, Tottori University School of Medicine, 86 Nishimachi, Yonago, Tottori 683, Japan, and ttThe Molecular Neurobiology Laboratory,The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaragi 305, Japan

We have isolated cDNA clones encoding an inositol 1,4,5-trisphosphate receptor type 1 (InsP3Rl) from human uteri and aleukaemic cell line, HL-60. Northern-blot analysis showed thatapprox. 10 kb of InsP3Rl mRNA is expressed in human uteri,oviducts and HL-60 cells. The predicted amino acid sequence ofhuman InsP3Rl (2695 amino acids) has 99 % identity with thatof the mouse SI-/SII- splicing counterpart. Western-blot analysiswith anti-(mouse InsP3Rl) antibodies showed that InsP3Rlprotein of human uteri and oviducts of approx 220 kDa isimmunostained. Northern-blot analysis ofHL-60 cell differentia-

INTRODUCTION

Inositol 1,4,5-trisphosphate (InsP3) mediates the effect ofreceptors linked to polyphosphoinositide (PI) hydrolysis onintracellular Ca2+ mobilization in a variety of cells [1]. Ca2+ is a

modulator of the physiological functions of the cells. InsP3selectively binds to a receptor that incorporates a Ca2+-releasechannel and is localized to the endoplasmic reticulum, which isconsidered to be an intracellular Ca2+ store [2]. Previously, our

group and others purified the InsP!3 receptors (InsP3Rs) fromrodent cerebella, and determined their primary structures bycDNA cloning [2-5]. The InsP3 binding sites and channel domainswere shown to be in the N-terminal and C-terminal regionsrespectively [6,7]. The remaining part of the receptor hasphosphorylation sites recognized by protein kinaseA (PKA) [8,9].and binding sites for ATP [3,5,10,11]. Protein kinase C [12] andCa2+/calmodulin-dependent protein kinase II [9,12] alsophosphorylate the InsP3R in vitro. Binding sites for calmodulin[10,12] and Ca2+ [13] have also been reported. Alternative splicing[6,14,15] has been considered to confer further functional com-

plexity. These studies suggest that InsP3R is a key molecule intowhich various kinds of signal pathways converge. Recently,cDNAs of distinct types of InsP3Rs have been cloned [16-18].Thus the original receptor is now named the type-I receptor(InsP3Rl) and is widely distributed in various tissues.We also isolated the cDNA of InsP3R from Drosophila

tion along the neutrophilic lineage induced by retinoic acid ordimethylsulphoxide showed an accompanying enhanced ex-pression of InsP3Rl mRNA. Immunohistochemical analysis ofthe cerebella of spinocerebellar degeneration patients showed avariable loss of Purkinje cells with an altered pattern ofimmuno-staining. The InsP3Rl gene (Insp3rl) was localized to the 3P25-26 region of human chromosome 3. The data presented hereclearly show that InsP3Rl exists widely in human tissues andmay play critical roles in various kinds of cellular functions.

melanogaster [19] and Xenopus laevis [20], and showed goodpreservation ofthis receptor among the species. Thus we assumedthat a similar InsP3R exists in human cells. There are indeedstudies showing that rapid formation of InsP3 and subsequentCa2l release occur in response to extracellular stimuli in humancells, such as smooth muscle cells [21], T-cells [22] and platelets[23]. Furthermore, PI-Ca2+ signalling could turn out to be linkedwith some specific human pathologies. Recently, the putativepathogenic gene product of the oculocerebrorenal syndrome ofLowe (OCRL) was shown to have strong homology with asoluble 75 kDa inositol polyphosphate-5-phosphatase [24],suggesting that an altered metabolism of InsP3 affects Ca2+signalling in OCRL patients [25]. Manic-depressive psychosis,characterized by swings in moods, is known to be well controlledby Li' administration, probably through its inhibitory action oninositol phosphate metabolism [26,27]. In these situations, charac-terization of human InsP3R would be informative.Our previous study showed that InsP3R mRNA is present in

the mouse uterus [28]. In the present study, we first showed thatanti-(mouse InsP3Rl) monoclonal antibodies (mAbs) couldcross-react well with the putative InsP3Rl in the human uterus.Subsequently, we isolated cDNA ofhuman InsP3Rl from uterinelibraries first by immunoscreening, followed by hybridization.The amino acid sequence deduced from the cloned cDNA showedvery strong homology with that of the rodent InsP3Rl. Forpreliminary characterization of human InsP3Rl, we studied two

Abbreviations used: InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; InsP3R1, type-1 InsP3R; InsP3R2, type-2 InsP3R; InsP3R3, type 3 InsP3R;RyR, ryanodine receptor; RyRl, skeletal muscle RyR; PKA, protein kinase A; PI, phosphoinositide; OCRL, oculocerebrorenal syndrome of Lowe; mAb,monoclonal antibody; RA, retinoic acid; DMSO, dimethylsulphoxide; ECL, enhanced chemiluminescence detection system; OPCA, olivopontocerebellaratrophy; ABC, avidin-biotin-peroxidase complex.

** Present address: Department of Biology, Faculty of Science, Chiba University, Yayoi-cho, Inage-ku, Chiba 263, Japantt To whom correspondence should be addressed.The nucleotide sequence reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI Nucleotide Sequence Databases under the

accession number D26070.

781

782 N. Yamada and others

systems for InsP3/Ca2+ signalling. We first chose HL-60 cells,originally derived from a patient with acute promyelocyticleukaemia [29], to examine the transcriptional regulation ofInsP3R1 during differentiation along the neutrophilic lineage.Secondly, we focused on cerebellar Purkinje cells which areabundant in InsP3Rl in both mouse [30] and human [311.Immunohistochemical analysis with the anti-(mouse InsP3Rl)mAb reveals that immunoreactive patterns of Purkinje cells havechanged significantly in spinocerebellar degeneration. In ad-dition, we mapped the human InsP3R 1 gene (Insp3rl) to thespecific locus of human chromosome 3 and discuss its relevanceto already mapped diseases and genes.

MATERIALS AND METHODSHuman tissue preparationNormal parts of myometrium were obtained from pathologicalsamples ofmyoma uteri. Oviducts were also obtained from them.The patients were in either their 30s or 40s without complications.These tissues were cut into pieces and kept at -80 °C until use.

Preparation of membrane functions and immunoblot analysisThe microsomal fraction (P3) ofmouse cerebella was prepared asdescribed [30]. Crude membrane protein fractions (P2 + P3) ofhuman uteri and oviducts were prepared as described [5]. AfterSDS/PAGE (5-10 ,ug/lane) [32], the proteins were transferred tonitrocellulose membranes (Hybond-ECL; Amersham), and

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analysed by the Enhanced Chemiluminescence detection system(ECL kit; Amersham) using the anti-(mouse InsP3R1) mAbs4C1 1, 1OA6 and 18A10 [30,33] as primary antibodies.

cDNA cloning and sequencingAll bacterial manipulations and cloning procedures were per-formed by standard methods [34] unless otherwise mentioned.Total RNA from human uteri and oviducts was purified intoPoly(A)+ RNA by oligo(dT)-cellulose chromatography oroligo(dT)-Latex [35]. The cDNA was prepared using randomhexamers as primers and ligated into the Agtl 1 vector (Stratagene)or the AZAP II vector (Stratagene). We synthesized two oligo-nucleotide primers corresponding to the upstream portion ofhuman clones 6Y and R62 (Figure la) by the phosphoramiditemethod using a DNA synthesizer (model 382A; AppliedBiosystems). The cDNA was then synthesized by using theseprimers and a You-Prime cDNA Synthesis Kit (PharmaciaLKB), and ligated into the AgtlO vector (Stratagene). The libraryin Agtl 1 was first immunoscreened [36] with mAbs 4C1 1, lOA6and 18A10 as described [3]. All the libraries were also screened byplaque hybridization using the 32P-labelled cDNA inserts ofmouse InsP3RI clones [3] and putative human InsP3R clones(5 x 105 c..p.m. per ml). Plaque hybridization and washing pro-cedures using the mouse probes were performed at 50-60 'C. Inthe case of the human probes, those were performed at 65 'Cinstead. The sequences ofpositive cDNA inserts were determinedfrom both strands as described [19]. Some parts of the humanInsP3Rl cDNA were also isolated from a library derived fromdimethylsulphoxide (DMSO)-treated HL-60 cells.

Northern-blot analysis of human uteri and oviducts

8000 bases Poly(A)+ RNAs (5 jug) from human uteri and oviducts and totalRNA (5 ug) from the cerebella of ICR mice (Japan SLC) were

R62 electrophoresed and blotted on to nylon membrane filters.Northern blots were probed by the 32P-labelled human clone 6Y(Figure la) as described [3].

Expression of the ligand-binding site of human lnsP3R1.LCa lThe 3728-bp cDNA fragment 81SBI (Figure la) encoding theLC82' channell 1190 N-terminal amino acids was ligated into a mammalian

____ expression vector pBactS as described [7]. A neuroblastoma/=___ glioma hybrid cell line NG108-15 was transfected with the

1| ,1iIII resultant plasmid--pB81SB DNA by the calcium phosphateprecipitation technique [37]. The expressed protein was expected-I__X 1 - [to appear in the cytosol. The preparation of the cytosolic proteinsand Western-blot analysis using mAb 4C1 1 were performed as

2500 described [7,19].

Figure 1 Human lnsP3R1 cDNA clones, functonal domains and hydro- lnsP3-blndlng assayphilicity of the deduced protein The assay were performed as described previously [7].(a) Schematic representation of human InsP3R1 cDNA clones from uteri. The open boxrepresents the protein coding region. Nucleotide residues (shown at the top) are numbered fromthe first residue of the 5' untranslated sequence. Solid bars indicate the clones used for thesequencing. (b) Functional domains and cysteine residues conserved among the InsP3R family.Human InsP3R1 consists of three main domains: an InsP3-binding domain, a modulatorydomain (V, PKA phosphorylation consensus sites; 0, putative ATP-binding sites), and aputative Ca2+-channel domain (six vertical thick bars, membrane-spanning segments M1-M6).Vertical lines beneath the open box indicate the conserved cysteine residues among the InsP3Rfamily. The longer lines indicate cysteine residues which are conserved even in the RyR. SI andSll denote the sites alternatively spliced out in the mouse InsP3R1. (V) show putative Asn-linked glycosylation sites. (c) Hydrophilicity plot (window size: 17) of the deduced humanInsP3R1 according to the method of Kyte and Doolittle [66].

Northern-blot analysis of differentiated HL-60 cellsHL-60 cells were cultured as described [38]. 1.25 % DMSO or1 juM retinoic acid (RA) were added to the culture medium for1-3 days to induce granulocytic differentiation. RNA washarvested with a Fast Track Kit (Invitrogen) and Northern blotswere probed by a 32P-labelled InsP3Rl cDNA of the HL-60clones [34]. The control probe was human fl-actin cDNA(Clontech). After autoradiography, relative quantification wasdone by scanning densitometry (CS-900OU; Shimazu).

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Figure 2 Western-blot analysis of InsP3R in human tissues

Membrane proteins from human uteri (15 /tg/lane), human oviducts (10 1sg/lane), and mousecerebella (1 ,ug/lane) were subjected to SDS/5% PAGE and were detected by immunoblotanalysis with anti-(mouse lnsP3Rl) mAb 10A6 and an ECL kit. The other two mAbs, 4C11 and1 8A1 0, gave essentially the same results (results not shown). The positions of molecular-massmarkers (in kDa) are shown at the left.

Immunohistochemical analysis of the cerebella of thespinocerebellar-degeneration patientsSamples from 17 histologically normal cerebella of humans(20-65 years old) and 16 cases of spinocerebellar degeneration[14 cases of olivopontocerebellar atrophy (OPCA) and two cases

of hereditary cerebellar atrophy of Holmes type] were examined.All the cerebella were obtained at autopsy within 6 h of death,fixed with 10% neutral formalin or 4% formaldehyde in 0.1 MPBS, pH 7.4, and embedded in paraffin. Sagittal and parasagittalsections (3 ,um thick) were initially stained by the haematoxylin-oesin, Kluver-Berra and Bodian methods, and the subsequentsections were examined by the avidin-biotin-peroxidase complex(ABC) method [39] using mAb 4C1 1 [30]. For control sections,the primary antibody was replaced with PBS.

Chromosomal mappingChromosome spreads were obtained from phytohaemagglutinin-stimulated human lymphocytes cultured for 72 h. 5-Bromodeoxyuridine was added for the final 7 h of culture(60 #4g/ml ofmedium), to ensure post hybridization chromosomalbanding of good quality. The mouse InsP3Rl clone, p23-2 [3],was 3H-labelled (2 x 108 d.p.m./,ug) and used as a probe. Theprobe was hybridized to metaphase spreads at a final con-

centration of 50 ng/ml of hybridization solution as described[40]. After coating with nuclear track emulsion (NTB2; Kodak),the slides were exposed for 20 days at 4 °C, and then developed.To avoid slipping of silver grains, chromosome spreads were first

Figure 3 Northern-blot analysis of lnsP3R1 mRNA

Lanes 1 and 2 contain 5 ,ug of poly(A)+ RNA from human uteri and oviducts respectively. Lane3 contains 5 ,ug of total cellular RNA from ICR mouse cerebella as a positive control. The arrow

indicates a putative lnsP3R1 mRNA of approx. 10 kb.

stained with buffered Giemsa solution and metaphases were

photographed. R-banding was then performed by thefluorochrome-photolysis-Giemsa method and metaphases re-

photographed.

RESULTS AND DISCUSSIONWestern-blot analysis of human tissuesWe first determined whether human tissues possess proteinsimmunoreactive with the anti-(mouse InsP3R1) mAbs. Pre-viously, we showed that InsP3Rl is located in smooth-muscle-enriched tissues of mice such as myometrium [28]. The crudemembrane fraction of both human uteri and oviducts containeda 220 kDa putative InsP3Rl protein which was immunoreactivewith all three mAbs (Figure 2). The protein has a slightly lowermolecular mass than that of the mouse cerebellar InsP3Rl(250 kDa), probably due to tissue-specific alternative splicing[15].

Isolation of human InsP3R cDNATo isolate human InsP3R cDNA from uterus, we constructedthree human uterine cDNA libraries (see the Materials andmethods section). We first immunoscreened the expression libraryby mAb 18A10, and isolated several positive clones includingclone R62 (Figure la). Subsequently, we obtained a full-lengthclone of putative human InsP3R cDNA by continual screeningsof the libraries with parts of the mouse InsP3Rl [3] and the

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784 N. Yamada and others

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Area 1HUM-i 1051 LHLTMHDYPPLVSGALQLLFRHFSQPQEVLQ)AFKQVQLLVTsQDVDNYKQI[KQDLDQLRSIVEKSELWvYK-GQGPDE7Hr,GASGENEHJCJTZE8GN-----KPQKHESTSSYNYRVMUE-i 1065 ........................................... ..P.........T -----S..L.......PAT-i 1065"........................................... ..P.........T-----S. .L.......XEL-i 1046 ........ .H................................-S.2.E.VTAAQA.GAD---KG.TP -----G.AJK.S......DOR 1168 ...I....A.....H ............P.SDS..ES ...S...I..QS ......-AKAT..LGATDA.GDA---VO_LEY------ AAkLSQ.QRN--E.. NRAT-2 1062 I..II....A..L......K....A........SN......A....LT.....E.S.SYENGD.GEGQAIKGG8EAN. .S.LLSPVQDGA.TPQID.NKGM.N.. I1PAT-3 1060 ............K.....AMHT......ISA... .8...V ..SE.P. .TMH.....D.K.SVKG.EGEAGASKDKKERPSDEE-------GL.P.GEK. .8..QlHUH-1I 1162" KIILKCQSSP-SXQRLNGHVLLQPEADXQIRAELNCGQNALIHNFNGLATQIMNQCENRVHuE-i 11716............................................ . . . . . .K.RAT-i 1176.............................................................XEL-1 '154 ..L..... NTG.--RN........ 2..S......T...R....KI.............. .T...............ORG 1274 .....M.F. .TA.GPGSV,V.P. .HE....V.V.T . ...D...N.DEKD.EL.K.L.C......L....V.. .N.LD......K.VCA. .KD.LA. .N.VTDK..PAT-. 1 82 ..... -----C.H.H. .....K.......D.....T-.E..N.V.D.. .T.P...P.P...V....L.... T. .L .. E.8. .P....YH..N.S.2...RAT-3 1172 G..0..N M.H.GV;E----QM..K..K.1..D..K.M.D....D.N-.N. .M..L.YT.Q.. .K.--....LQO .. T.L.L...E....L..Y.... P.L.

HUM-1 1280 CEHPVYKLTVAGFKKQ*4EVSEVVYDAFTIQMS--EDMESLtYILELACEKVTICSLLDVVHUE-i 12194.............................................................RAT-i 1294......................................... . . .F.XEL-i i2172 . .A............. PY....I. .V...........---...... E... -......................DOR i393.....I....H.A.LQ. .... V.A..NQ. .PP....Q.I........KG..NHFV. .. .QQQHLGHEKLSDD...-K. .VE. .K .. C.M.H......N.S.2...TIIPAT-2 12195.......H.E.LR......n. YV.T....T..I.G...I.....PI.LN. .C.---. .A.G. .S0..-A. .. T....A..............PAT-3 i2815. H.P.....H .. .LD. .H.I....YV....I.T. .T.A.D. .V....K. .LAH.LD. .KA---A. .GVEDH...-.. ...S.0D...A.A.......T....E.V-S..

Figure 4 Alignment of the deduced amino acid sequence of human InsP3R1 with those of the mouse, rat, Xenopus laevls and Drosophila melanogaster

The predicted amino acid sequence of human InsP3R1 (HUM-1) is shown in comparison with the published mouse type-1 (MUS-1) [3], rat type-1 (RAT-1) [4], Xenopus type-1 (XEL-1) [20], Drosophila(DRO) [19], rat type-2 (RAT-2) [16], and rat type-3 (RAT-3) [17] sequences. Identical amino acids are indicated by dots. Dashes indicate gaps (deletions or insertions) for maximal homology.

Human inositol 1,4,5-trisphosphate type-i receptor 785

MUN-1 1396 THEDCIPEVI(IAYINFLNHCYVDTEVEPO(EIYTSNHMwKLFE-NFLVDICRACNNTSDRICHADSILEKYVTEIVMSIVTTFFSSPFSDQSTrLQ------TRQPVFVQLLQGVFRVYMUS-1 1410.............................................................RAT-1 1411 ................................ . . .V............................XEL-1 1388...v................V........V..S .....!V..R ......N.........ASLAVVQ.......AlT....DRO 1512 C.PL.M. .....E..VD....I......A.G.. .S .. .KS....NQLIT.PAA--ASNKT.QA. .LNG.TWLLGS. .A.....AIV.------S. .LI....AAH.ITRAT-2 1411 ..D......V..V...........I.......M.V..T.T....TF..RC...S..N..SG..N....N..S..--------H..I..S.IRAT-3 1401.T...T...M..V. .V...........I.T. . .... TL.MALV.-.KREKRLS.PT...LTV.LDTISA.....EN. .S. --------H.TIV....STT LL

MUS-1 1520.-........................N....L. ---...............................RAT-i 1521 ......................... .-N....................................XEL-i 1507....L............T.-..........N..S..S.M.V..---.......T..........S..............DRO 1621 Q.R. .SLG2DRfN. .N_...T.TES. .M.S. .L.PE.EQ. .ATMSS.T-AMLTRQ.TKWLLA.KQPKYE---AQQAASLM.WD.S. ...G.....L.. Q.K.V.E... .L....I.Y.S...RAT-2 152' N.T.PN.A......A.AE. .. .N.G.......T. .M.N.S.T. .RA. .G....SGP.-F-KEALGGPAW..K...X..V.AS. .QQFS.1M.4. .F.....YS....RAT-3 1510 E.-P. .QQH.G....A.V.T.AN.....LL.MN...AIMSA.LSSGGS--CSAA.QRSAANIYKT.T.TFP.VIPT.NQW. .K... .K.....IT .. .E..K....... W..

NUN-i 1622 FPENTDARPKCESGGFICKLIKHTKQLLEENEEKL.CIKvLQTLREMMTKDRGYGEKX---------------------GEALRQVLVNRYYGNVRPSGRRESmus-i 1636..............................QISIDESENAELPQAPEAENSTEQELEPSPPLRQLEDHKR....I.....I.....RAT-i 1637 ..Q.......................... ISIDELENAELPQPPBAENSTE-ELBPSPPL.RQLEDHKR....I.....I.....XRL-1 1623.....K....Q....R ...L............A.... F.D.------------------------VI.I.... ..A...DRO 1737 . .AG.E. .KR ....R....EKX . . ..X.RM.V. ...R....AI.VN.------------------------D.0....T.LL. .FQTSTPRLP.DRAT-2 1639 . ..GS. .. .IR.--.A.MS....N. ..K.M.- .....I.....LE.KDSF'M.E ---------------------SST. .KI.L. ..FKGDHSV.V---RAT-3 1628 .L.GSE.YQR....LS. ..R.-..G.M.-S....V....R..QQ.LQ.KSK. .DR------------------------- M.LQN.LQ.-.K. .P.--

Area 2 ..

HUN-1 1702 LTSFGNGPLSAGGPGKPGGGGGG-SGSSSMSRGEMSLAEVQCHLDKEGASNLVIDL- IMNASSDRVFHESILLAIALLEGGNTTIQHSFFCRLTEDKKSEKFFKVFYDRMKVAQQEI]KATMUS-1 1756......P... ..-....P... .T..............................................RAT-1 1756......P...S.-.....P.G.T..............................................XEL-1 1703 ......G.SS. .--S. .. .S-I. .G.L.S.....SD ..Q..Q.. ..-...... T .......................F.......DRO 1817 EVPLLAA. .IDPAKQN HLVTH.-P.AKYLQ.AGKT.H.M.N. ... .....D. .VE.V.KSVH.PNI.V.AVE.G.....PI..KGM.QKFLS.DL.NQA....FEK.D.....S.RAT-2 1713-.......AYA.TAQV. .. .PT.QDADKT.-I.MSDI. .L.....E... .-V.V.TKN. .I.S.G.. .G.... T. .N. .YQQ.H.Q......L....A. .K. .RS.RAT-3 1704-- .--E.----TD.T.S.VD-O2W -----AI.AT...8. TI..T.0.C.-.TSTKNEKI.Q... .G...R.D.D.. . E..K. .YNLM.S...R.....LH ... R.R. ...T.S.

HUM-i 1820 VTVNTSDLGNKKKDDEVD-R----DAPSRKKAKEPTTQITEEVPDQLLEASAATRKAFTrF'RREADPD-DHYQPGEGT -----QATADKAKDDL-EMSAVITIMQPILRFLQLLCENRAT-i 1874..................................-.. ..s...S--------- T.T.................XEL-1 1819......S. .R.E.QSE.----ET.HHQRVR. .SG...AKE...1...V. .K. .YYS........FSL....V-----M.V.E.GR.E.-............DR0 1935....T.IAA.AHiEHKQ.TNLELDKIARKHG;L.SNMGVV.... LKRE.HN.GL. .AR.YGNA.NIHSGEESSAISVNSPLEDILAEKLEKHKDSR.QRN4QL.NKVLV........RAT-2 1825 ...I....S. .REEDS.LM----ALGP.?mRVRDSSLHLK.GMK(G..T....S. .S. .YCVY...M.M...I.TMC. .QEA-----GS.EEKS.EEV--T. .PA. ... .R.......RAT-3 i804 .A. .M.....SQPRE----- EP--AD.TT.GRVSSFS------NP-.SSRYSI,GPGLH.GH.VS-ERA.NN --------------GTSVL.R.8......

MUS-1 1979.............................................................RAT-i 1979.............................................................XEL-1 1925..........S.. . . . .........................................R .DRO 2055 . .P.M. .L..N.N...N ...S ..M............N.L.....A.............L......N....EN..........RAT-2 1932 ....E....N......................V...........T........I....S.....Y.Q .RAT-3 1883................IMN..........D.G.VI. . ...T..T.....T.V ..........S..C.Y ....Q.D.0...:L,HUM-i 2045 MESRHDSENAE.RILYNMRPKELVEVIKKAYMQGE-----VEFEDGENGEDGAASPRNVGHNIYILAHQLARHNXELQSM.LuPGGQ-----------------Vmus-i 2099..........................................T....G-----------------RAT-i 2099.......................................... T.-------------------XEL-1 2045..............L...L---------- -S...Y..............H....V.T ----------------GDR0 2175 .... r.G..N....N.Q. .....AC .. H.E.LIDEQDDGD.PDA.SDDD.ATV. .. .E.....C... .Q....AGL. .ASBDPQSASFD -------------ARAT-2 2052 ........F.....D.M.N. .N. .-L-----ECNHGD.E.G.DGV.K..1....L.....L.Q....SDP ----------------ERAT-3 2003 ........ISL. .Q ... .....L.E.----- .---R----NSEV. .. .E......L..S.. ..Q. .HL. .. .VKRIQEEEABGISSMLSLNNKQLSQMtKSS"pAQE

M]HUM-i 62126 YKTQI RLRMQ PP EFT R YTRDQ S DFRSE LWMWK RAPLWANSFWSISFIVMNLAFP-KMUS-1 2180.............................................................RAT-i 2180.............................................................XEL-1 2127E.. . . .H............................H...Q..........Q..S...T.......-......VNDR0 2271 KTSQ. .MY. .T......N.. .L....I.E....Y..TDT.IK.J.N.A... D....L.A .. .DKAEM.....K....S..L.F.ISSY. .L. .N.L. .CV.VI.MI.....-ODNRAT-2 2134 E.K..Y. .N....H...M......N.....R..Y.VFN......V....QQT. ...Y ...K.....I.M.A.F.FS.HI.L.G.....F.F.-A. .L .... GDDRAT-3 2108 EEEDP.AY.EN. .S..Q..Q.S......A.. .Q.. E.T.H.LFTr...Q....VS.. .DQ.SF.H. .. .E..RR..SM.LI. .FS.R.TL.G.....FI.jIII....VE.

M2 M3 M4HUM-i 2245 VRGLEHSL-THILIILKHIAISIRISGQTFLAMNIFMFGCTTGRMLVFYLYVCNLVEFSLNUs-1 2299 ......................................................L.........RAT-i 2299 ......................................................L.........XEL-1 2246 .H. .... -DSRL . . ..-...V.V.............I............T..8..R....G.........L...V......080 2390 T-VPE.-SS.I.-. .F.IITIF. .V. .L. ..RES. ..TF.G.V. .. .F..LL.PES. .C.. .VVT.TL.SVHIV.IM. .K. .LEKQLIKIIT.FSTYTA.YSVLL--LR.IF.P....RAT-2 2254 GDE. ..-S.LF.A. .-.V.VA.CTSMLFFFS. .V....PFLV.IM. .S.YTI. .G..I.1....A.L... V..V..V.R......VI. .MA.... VA.VLV.MI .......F..RAT-3 2228 AST.V.GS.LI.-._.F.ILICF.I.ALF--T.HYSV.P. .VALV. .S.YYL.IG. .. .NI ...L.LT....V.VV.R..R.. I....K ..N.M-M....VG.ILTSVL... A.L.L.. I1..

M5 Area 3.HUM-i 2363 FDLVYREETLLNVIKSVT NrGRSI I TVA VLSIV L KDFILVRLN TVEGESLSEFF VRESG-ECSPP VAEED T TLMUS-1 2417 ......................................ND.Y.Y....T.-... .r.. .K.i...L...........RAT-i 2417 ............P..A...............-......G.....ND. .Y.....T.-...T.....K....L.V...........XEL-i 2364 . ..............................Q.--. .IF. .N.GThT. .L.YPE. .... G.D-T. .TH---E.LAQVT. .E.EE.-....DRO 2505 . .V.....V..R......V.I.......I.M....LVS ..FEEQ--DN.P.PSVPLTL.VPVSG.S.SAPDDLG. .QAA--K.V"P.SAGGGEV.-.RS.DiS.V'RAT-2 2372 .....I..F.....L................K.--R.P.TGNDGVPT-MT.T.MLGTCPK-......-..T1PSSNA-.G.GGE.GI.R.D.....RAT-3 2345 ....1....F........L....L..F.....F.L.......GNHSR.STLGMPHG.AT.M--GT.SGDK--MD.V.BVSVP.IL-E.DE-L.ST.RA.D ..

M6HUUM-i CIVT2479GVGDLKPX PFA DLFFVIIIVNLIFVI TADRSEQKEE KT ICLE KDKVFEHX ENWYC LKKDMUS-1 2533.............................................................RAT-i 2533.............................................................XEL-1 2477.....................................V...........A...F......F......DRO 2620 . ....T.NQ. .. .N.. .I..I. .A. .SK.G. .V.......I.Q............Q.A......S.M.SA....S....S.....Y..... PRAT-2 2485 ....NQ ...N......R..D......V.....I...............K............S....S.....Y..... PRAT-3 2460.... MN ....N....I ..0.5.DS.P...V.....I..........................S....L.-N.Y.Y..8.R.NKHUM-i 2599 TETPSVEIENDFRRMLSDEENLNQKETKVNSQSLDMERQQILGPHNNQPMUS-i 2653 ....... P......L......................................RAT-i 2653 ....... 8...........................................XEL-1 2597..........D...................P................8..Q.A.L.1....080 2740 . .F.....YA.V.AG1.E. -. .L...AAV.AD.. .I.. .SM.AQ.LD.QL.IKF. .T. .H. ... H..L....L.. MNTANSLLPF.RAT-2 2605.......Q.T.(.........NEGDS. .... I..S.....S.KQ....A..E.E.N... .--L.F. .SNTPNENHHMP.HRAT-3 2580 D.0....Q....NK.........GEG....1.1..7...:....SH.TA.N.M...E.....RR. .L.FVDVQNC.SR

The predicted transmembrane regions are marked by solid lines and denoted as M1-M6. Consensus ATP-binding sites are marked with bold broken lines. Bold lines and filled triangles representconsensus sequences for PKA phosphorylation and possible phosphorylated Ser residues respectively. SI and Sll are regions that could be deleted by alternative splicing in the mouse InsP3Rl[15]. Large filled circles represent putative Asn-linked glycosylation sites. Areas 1-3 are where sequence diversity among the lnsP3R family is prominent.

786 N. Yamada and others

putative human InsP3RI cDNAs. One cDNA clone was lesshomologous with the mouse InsP3Rl, and corresponded to thetype-2 InsP3R (InsP3R2) [16].The complete cDNA sequence (9076 nucleotides) was de-

termined by sequencing the six uterine clones shown in Figurel(a), and included a single long open reading frame (nucleotidepositions 257-8341: an open box in Figures la and lb) precededby an untranslated leader sequence of at least 256 nucleotides.The surrounding sequence of a putative initiation codon,GAGATGT, was in relatively good accordance with Kozak'soptimal sequence for translation initiation [41], although anotherin-frame methionine-coding codon (nucleotides 269-271) mightbe used as a translation initiation signal. We could not find anypoly(A) tracts in uterus-derived clones. There was an adenylationcontrol element (UUUUUAU) assumed to be involved in thecontrol of translation [42] 89 nucleotides upstream of thepolyadenylation signal. We also isolated six cDNA clones for theInsP3Rl from a cDNA library of DMSO-treated HL-60 cells.From these clones, we could determine the sequence of 4387bases including the complete 3' untranslated region and a poly(A)tract. The 5' 3932 base sequence of the cloned myeloid cDNAcompletely matched with the 3'-terminal sequence of the uterinecDNA. Thus the total length of cDNA isolated was 9531nucleotides long. The complete 3' untranslated region of HL-60cells contained one polyadenylation signal, while that of themouse InsP3Rl [3] had two. Northern-blot analysis (Figure 3) ofhuman uteri and oviducts showed that the cloned cDNA couldhybridize with single RNA of approx. 10 kb, which agreed withthat of the mouse InsP3Rl mRNA [3].

Sequence comparison among the insP3R familyThe human InsP3R deduced from the cDNA sequence wascomposed of 2695 amino acids with a calculated molecular massof 307 kDa. The amino acid sequence of the human InsP3R has99% identity with the sequences ofboth mouse and rat InsP3Rls.Mouse and rat InsP3Rls share approx. 70% and 62% identitywith the rat type-2 [16] and type-3 [17] receptors respectively.Therefore, our human clones could be InsP3Rl. Furthermore,the present human receptor was categorized as one of thealternative-splicing subtypes of mouse InsP3Rl [15], lacking twosplicing segments SI and SII (SI-/SII- subtype) (Figure 4). Oursequence data on SI1 splicing was consistent with the previousobservation that the SII+ subtype exists exclusively in neuronaltissues [14,15].

It should be noted that Ser-666 is a human-specific insertion,and is not found in the corresponding site of InsP3Rls fromother species (Figure 4). Among 32 amino acid replacementsbetween human and mouse InsP3R1, 17 replacements concentratein three areas. Four replacements concentrate in Area 1 (Thr-1 128-Gln-1 149: 22 amino acids), six in Area 2 (Val-1684-Met-1730: 47 amino acids), and seven in Area 3 (Ser-2434-Val-2460:27 amino acids). These areas are also consistent with the variableregions among the InsP3R family (Figure 4). The sequencesimilarity between the mouse InsP3R1 [3] and the rabbitryanodine receptor (RyRI) [43] is also observed in humanInsP3Rl (e.g. eight conserved Cys residues: Cys-623, -1231,-1922, -1993, -2473, -2479, -2556, -2559 in human InsP3Rl,Figure Ib).

Transmembrane topology and the putative Ca2+-release channel regionHydrophobicity analysis of the human InsP3Rl protein (Figureic) revealed several hydrophobicity stretches which might

2222-2535). These putative membrane-spanning sequences clus-ter around the C-terminus (Figure lb) and, together with theneighbouring sequence, might constitute a Ca2+-release channel.From previous studies [3,19,44], we have proposed that InsP3Rtraverses the membrane six times. Therefore, we assigned thenames M1-M6 to these six sequences (Ml, residues 2222-2240;M2,2254-2272; M3,2298-2317; M4,2337-2353; M5,2386-2408;and M6, 2516-2535). M1-M4 are less conserved among thewhole InsP3R family than M5 and M6 (Figure 4), which arehomologous even with the RyR family. Of eight cysteine residuesconserved in InsP3Rs and RyRI described above, four cysteineresidues are present in this region (Figure Ib), suggesting theexistence of a common mechanism to maintain or regulate thereceptor-channel conformation. In the N-terminal half of theputative third intraluminal loop (between M5 and M6), there aremany acidic residues and putative N-glycosylation sites (NET,2421-2423; NCS, 2449-2451) in addition to Area 3 (Figure 4).We recently confirmed that the mouse InsP3Rl is glycosylated atthese corresponding positions in an Asn-linked manner[44a]. Interestingly, human InsP3R2 has two consensus sites inthese corresponding positions, while human InsP3R3 has one[44b]. The function of these sugar chains of the InsP3R familyremains obscure. The cluster of acidic residues seems to beinvolved in efficient Ca2+ permeation by concentrating Ca2+ nearthe putative channel pore [19]. In spite of the sequence variabilityin Area 3, these acidic residues (Asp and Glu) are strictlyconserved among the InsP3Rls. Thus the stretch could constitutean integral part of this type of Ca2+ channel. The sequencedivergence in the regions such as Area 3 in turn may be relatedto the distinct channel properties among the family.

Putative InsP3-binding regionPreviously, we showed that the 650 N-terminal amino acids area prerequisite for InsP3 binding [7]. The amino acid sequence ofthe corresponding region of human InsP3Rl (Met-l-Glu-635) isalmost identical with that of mouse, except for SI deletion andtwo conservative amino acid substitutions (human/mouse, Thr-43/Ala-43; Val-408/Leu-423). In and near this region, 11 Cysresidues are conserved among the InsP3R family (Figure lb:Cys-56, -61, -206, -292, -379, -541, -623, -638, -730, -753, and-768). In addition, it was reported that mutation of Arg-615 ofRyRi to Cys has been identified as the possible cause of pigmalignant hyperthermia characterized by hypersensitive gatingof RyRI [45]. This mutation of RyRI is thought to lead toabnormal regulation of the channel gating by its ligands andmodulators. There is 31 % sequence identity between the 30amino acids surrounding Arg-615 of RyRI and human InsP3Rl(note that Cys-623 in the corresponding region ofhuman InsP3Rlis also conserved in RyRI: Figure lb). This homologous regionis therefore a candidate site concerned with the fundamentalarchitecture for the binding of analogous ligands and/or for thecoupling of ligand binding to channel opening.

Modulatory regionThe vast region between the putative InsP3-binding region andthe transmembrane region, has been designated as the modu-latory region. Two consensus sites for PKA phosphorylation inrodent InsP3Rls [3,4] are conserved in human forms also(RRDS/1571-1574, RRES/1698-1701). The rodent InsP3Rl hasthe neuronal tissue-specific SII splicing segment (40 amino acids)between these two potential phosphorylation sites [15]. Thehuman InsP3Rl cloned from uterus and HL-60 cells does nothave this SII segment at the corresponding position (betweenrepresent multiple membrane-spanning sequences (residues

Human inositol 1,4,5-trisphosphate type-i receptor

residues Lys-1677 and Gly-1678), although the neuronaltissue-specific SII+ subtype has not yet been found in human.Thus the present human SII- subtype could be phosphorylatedmainly at Ser-1574, according to previous work which showedthat the splicing segment affects the alternative of two PKAphosphorylation sites in rat [14]. Two potential ATP-bindingsites (GXGXXG) have been found in rodent InsP3RI [3,4]. Oneis well-conserved among the InsP3R family (ILGLLG;1962-1967 in human InsP3R1). The other site is in Area 2 wherethe amino acid sequence is divergent even among InsP3Rls, andis composed of two overlapping consensus sequences in rodentforms. A replacement (human/mouse, Gly-1716/Ser-1770) ofthis area adds one more overlapping consensus sequences(GPGKPGGGGGGSG; 1714-1726) to human InsP3Rl. Be-cause of the amino acid sequence diversity, the later consensussequences are absent in Drosophila InsP3R, and rat InsP3R2 andInsP3R3. Removal of the SIT segment creates an additionalconsensus sequence for ATP binding in rodent [46] and humanSII- InsP3Rl (GYGEKG; 1673-1678). Thus the alternativesplicing might control patterns of ATP binding as well as PKAphosphorylation of InsP3Rl, resulting in modulation of channelactivity [10,1 1,47,48,48a]. It is known that the ATP binding tothe purified cerebellar InsP3R1, which is predominantly the SII+subtype [15] without the additional ATP-binding consensus site,has a stoichiometry of one [10]. Therefore, the actual ATP-binding site and its physiological role may vary depending uponcell types. There is a cluster of 13 cysteine residues (Cys-1 173,-1231, -1270, -1284,-1312, -1370, -1382, -1400, -1415, -1507,-1521, -1632, -1657) in 484 residues (1173-1657) which is strictlyconserved in the InsP3R family (Figures lb and 4). OnlyCys-1231 is conserved in RyRI. Around this region, there isthe longest stretch of non-homologous sequence (Cys-1405-Ser-1736 in human InsP3Rl) between InsP3R and RyR [3]. Itwas recently proposed that InsP3Rl has distinct classes ofcysteine residues with different sensitivities to thiomerosal, andthat modification of specific cysteine residues by the reagent canalter InsP3-induced Ca2+ release without affecting Ca2+-bindingactivity [49]. Thus some of the cystine residues in the modulatoryand channel regions may take part in the regulation of Ca2+-channel activity of InsP3Rl.

(a)

o

kDa

E-

a)0

,a)0

O0

200

116-97.4-

66.2 -

(b)

500 -

400-

C~

C-

a. 300 -

cQ0 200 --

Q00u)1 o n=2

Vec

Figure 5 Analysis of expressed human insP3Rl cDNA clone, 81SB1

(a) Immunoblot analysis of truncated protein of human lnsP3R1 expressed in NG108-15 cells.Cytosolic proteins from NG108-15 cells transfected with the truncated construct DNA pB81SB(10 1ug/lane) and vector DNA pBactS (10 zg/lane) as control and mouse cerebellar microsomalfraction proteins (1 1ug/lane) were analysed by immunoblotting using mAb 4C11 and an ECLkit. The positions of molecular-mass markers (in kDa) are shown at the left. (b) Specific bindingof [3H]lnsP3 to the expressed protein. 81 SB1, soluble proteins from NG108-1 5 cells transfectedwith the truncated construct of human lnsP3R1. Vec, soluble proteins from NG108-15 cellstransfected with vector DNA as control. N, number of samples. The data are the means of nsamples. Specific binding was defined as the total binding minus non-specific binding asdescribed [3,7].

*nsP, 10 b.lnsP3R =^ 10kb

Actin

StimulusTime Idays)

Relative lnsP3Rexpression

- 2 kb

- D R D- 1 1 31.0 3.2 3.5 2.3

R34.4

InsP3-binding activity of expressed receptor proteinsTo confirm the InsP3-binding property of our cloned humanInsP3Rl, we transiently expressed the clone 81SB1 (Figure la)encoding the 1190 N-terminal amino acids in a neuroblastoma/glioma hybrid cell line, NG108-15. We first confirmed itsexpression by Western-blot analysis on cytosolic proteins usingmAb 4C 1. The cDNA-transfected cells produced a 130 kDaimmunopositive protein as expected (Figure 5a). The expressionof the truncated human InsP3Rl was apparently coupled withthe elevation of [3H]InsP3-binding activity (Figure Sb). Thevector-transfected NG108-15 cells exhibited almost no increasein InsP3-binding activity. Therefore, our cloned human uterineInsP3RI has InsP3-binding activity in the N-terminal putativelarge cytoplasmic region.

Northern-blot analysis of dMerentiated HL-60 cellsTo evaluate the expression pattern of InsP3RI gene during celldifferentiation, we used HL-60 cells whose differentiation towardthe neutrophilic lineage is induced by RA or DMSO [50]. Inundifferentiated HL-60 cells, a probe for human InsP3Rl detecteda single 10-kb mRNA species in Northern-blot analysis (Figure

Figure 6 Northern-blot analysis of lnsP3R1 mRNA in HL-60 cells beforeand after differentiationPoly(A)+ RNA was harvested from undifferentiated cells (left hand lane) or from cells exposedto either 1.25% DMSO (D) or 1 ,uM RA (R) for either 1 or 3 days as indicated. The lnsP3R1and 8-actin probes hybridized to 10- and 2-kb species respectively. Relative lnsP3R1 expressionis based on scanning densitometry and is corrected for the actin signal in each lane.

6). In HL-60 cells, induced to differentiate toward the neutrophiliclineage by either RA or DMSO, there was a substantial increasein the level of InsP3Rl mRNA. When normalized by an actinstandard, the range of the increase in InsP3Rl mRNA relative toundifferentiated cells was 2.3- to 4.4-fold. Sequence analysisrevealed that the transcript might be that of InsP3Rl, althoughother types can co-exist. The increase in InsP3Rl mRNA goesalong with the acquisition of functions such as phagocytosis,degranulation and enhanced oxidative metabolism which areknown to be associated with InsP3/Ca2+ signalling. The extent ofthis upregulation was somewhat greater and its time course moreextended with RA than with DMSO. The difference may reflecttheir distinctive ways of inducing differentiation [51]. Bradford etal. [52,53] recently reported transcriptional upregulation of the

787

788 N. Yamada and others

(a)

(c)

Figure 7 Immunohistochemistry of cerebella of normal humans and spinocerebellar-degeneratlon patients with antl-lnsP3Rl mAb 4C11

Original magnification x 50 for all sections. The nuclei of the granule cells which are not immunopositive are counterstained with haematoxylin-eosin to enhance photographic contrast. (a) Purkinjecells in a 64-year-old man with a histologically normal cerebellum showing specifically immunostained cell bodies, dendritic arborization and axons (arrowheads). (b) Purkinje cells in a 66-year-old man with cerebellar atrophy of Holmes type, showing unstained spiny branchlets and intensely stained cell bodies and main dendritic trunks. (c) Cerebellar cortex from the same case as(b) showing completely unstained Purkinje cell bodies (arrows) and an intensely stained cell body and main dendritic trunks. (d) Cerebellar cortex in a 64-year-old-man with OPCA showing aweakly stained Purkinje cell body on the right and an intensely stained torpedo (arrow).

InsP3R gene in HL-60 cells exposed to either RA or 1,25-dihydroxyvitamin D3 by using a mouse InsP3R probe. Aprevious study detected an increase in functional InsP3 bindingin cells exposed to either RA or DMSO [51]. Our data obtainedwith human InsP3R1 probes are consistent with those results andsuggest that the complex regulatory mechanism of InsP3RlmRNA expression could exist in the cells. In addition, it was

recently shown that InsP3-sensitive intracellular Ca2+ storesregulate Ca2+ influx in HL-60 cells [54,55] and that the capacityof the Ca2+ signalling system composed of Ca2+ release and influxappeared to increase during cellular differentiation [55]. Thus theupregulation of InsP3R1 transcription in the neutrophilicdifferentiation may reflect the increase in the overall capacity ofthe Ca2+-signalling system.

Immunohistochemistry of human cerebellaIn rodents, InsP3Rl is located predominantly in cerebellarPurkinje cells [3,56,57]. It is known that there are various mousecerebellar mutations like Purkinje cell degeneration, nervous,Lurcher and staggerer that show massive degeneration or markedunderdevelopment of Purkinje cells [58], causing the reduction inInsP3Rl expression [3]. InsP3Rl mRNA is also observed inhuman cerebella by Northern blotting (results not shown). Toestablish the localization of InsP3Rl in human cerebella, we

performed immunohistochemical analysis of normal and ab-

normal cerebella of humans using mAb 4C1 1, which reacts withhuman uterus and oviduct InsP3R1 (Figure 2). In normalcerebella, Purkinje cells were predominantly immunostained.The cell bodies, dendrites and axons of the cells were specificallyimmunostained (Figure 7a). The dendrites were clearly visualized,not only their main dendritic trunks but also the spiny branchletsstudded with numerous spines. The axons of the Purkinje cellswere traceable to the dentate nucleus, where the presynapticterminals of the axons were seen as immunopositive productsaround the immunonegative cell bodies and dendrites of thedentate nucleus neurons. In the cerebella of the spinocerebellar-degeneration patients, the most striking feature was that spinybranchlets of the Purkinje cell dendrites were not stained,although atrophic cell bodies and main dendritic trunks were

intensely stained (Figure 7b). There were some Purkinje cellswhose cell bodies were completely negative for the InsP3R1(Figure 7c). Purkinje cell axons and torpedoes were more

intensely stained than the cell bodies (Figure 7d). Our previousstudy suggested that the mRNA of InsP3Rl might well exist inthe dendrites of Purkinje cells [56]. A limited number of othermRNAs were shown to be present in dendrites [59,60]. Thedendritic localization may permit the local regulation of theirtranslation, probably including regulation by synaptic activity.Therefore, the patchy distribution of InsP3R1 protein observedin the cerebella of those patients may reflect the loss of suchlocal regulation. These findings demonstrate that InsP3Rl is

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potentially important in normal brain physiology, such aslong-term depression in cerebellum [61] and in aetiology ofspinocerebellar degenerations such as OPCA and hereditarycerebellar atrophy of Holmes type.

The InsP3R1 gene (Insp3rl) maps to human chromosome 3In the 120 metaphase cells examined, there were 254 silver grainsassociated with chromosomes and 71 of these were located onchromosome 3. A large percentage (78.8 %) ofthem were mappedto the p25-p26 region of the chromosome 3, with the maximum

in the 3p26 band (Figure 8). These results allow us to map theInsP3Rl gene (Insp3rl) to the 3p25-p26 bands of the humangenome. Comparison with chromosomal mapping of the mouse[56] reveals that the c-raf-J gene (3p25 in the human genome [62])exists in the vicinity of the InsP3R1 gene in both species. Theshort arm of chromosome 3 is one of four regions of humanchromosomes where loss of heterozygosity often accompaniesmany types of tumours, suggesting the existence of tumoursuppressor genes in the regions [63]. The genetic locus (3p25-26)is also coincidental with that of von Hippel-Lindau disease [64],the disease gene ofwhich behaves as a typical tumour suppressor.Recently, a putative tumour-suppressor gene of the disease wasisolated and partially sequenced [65]. We compared the deducedamino acid sequence of that gene with that of human InsP3R1,and could not detect any homology between the two genes.

We are grateful to Professor Ichiro Nagata and his colleagues (Division ofGynaecology, National Defence Medical College Hospital, Saitama, Japan) forproviding pathological samples and to Professor Daniel Lew and his colleagues(University of Geneva, Geneva, Switzerland) for support of the HL-60 cloning work.We also thank Mr. T. Michikawa for his help with the InsP3-binding assay. This workwas supported by grants from the Ministry of Education, Science and Culture ofJapan, the Human Frontier Science Programme, the Ministry of Health and Welfare,the Toray Scientific Research Foundation, the Senri Life Science Foundation, UnitedStates Department of Veterans Affairs and the Zyma Foundation.

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Received 1 February 1994/18 March 1994; accepted 28 March 1994